Many ongoing research programs focus on the development and utilization of new genomics and proteomic tools for studying cellular function. In contrast, relatively few research programs seek to analogously address the metabolome. The ability to temporally and spatially address the presence, identity, concentrations and flux of metabolites, small molecules and second messengers in live cells, tissues and organisms would have wide applicability to the study of basic biological and biomedical problems. Such abilities would play a major role in understanding the underlying pathways involved in cellular homeostasis, development and aging. Existing approaches predominantly are highly-invasive, toxic and lack the desired sensitivity, specificity, temporal resolution and sub-cellular targeting. Furthermore, most approaches are specific to a particular analyte and are not easily generalizable. This proposal seeks to develop a versatile platform for developing protein biosensors for identifying and quantifying cellular metabolites and second messengers and their fluxes at high anatomical, spatial, and temporal resolution in situ. We propose to create novel protein switches that couple small-molecule levels to beta-lactamase enzyme activity and to protein fluorescence. This will be accomplished through the in vitro recombination of existing proteins with the perquisite small-molecule binding and catalytic/fluorescent properties by a technology recently developed by the PI. Specifically, we plan to develop biosensors for Zn(ll) as a proof of principle of our approach. In addition to its structural and catalytic roles, zinc plays diverse roles in biological processes including the regulation of enzymes, the regulation of gene expression, apoptosis and cell-to-cell signaling. In addition, abnormal patterns of intracellular Zn(ll) accumulation have been found in patients with Alzhemier's disease, diabetes and cancer. Despite its diversity of roles, its importance has only recently become clear and the mechanistic details surrounding intracellular zinc accumulation, trafficking and function are poorly defined. Although we propose to develop sensors for Zn(ll) that will have important applications for understanding zinc's role in biology, the broader and more significant goal is to demonstrate our general strategy for making sensors for live cell fluorescence imaging of small molecules, metabolites and second messengers. [unreadable] [unreadable]